AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses

Abstract

Although a causal relationship between Zika virus (ZIKV) and microcephaly has been established, it remains unclear why ZIKV, but not other pathogenic flaviviruses, causes congenital defects. Here we show that when viruses are produced in mammalian cells, ZIKV, but not the closely related dengue virus (DENV) or West Nile virus (WNV), can efficiently infect key placental barrier cells that directly contact the fetal bloodstream. We show that AXL, a receptor tyrosine kinase, is the primary ZIKV entry cofactor on human umbilical vein endothelial cells (HUVECs), and that ZIKV uses AXL with much greater efficiency than does DENV or WNV. Consistent with this observation, only ZIKV, but not WNV or DENV, bound the AXL ligand Gas6. In comparison, when DENV and WNV were produced in insect cells, they also infected HUVECs in an AXL-dependent manner. Our data suggest that ZIKV, when produced from mammalian cells, infects fetal endothelial cells much more efficiently than other pathogenic flaviviruses because it binds Gas6 more avidly, which in turn facilitates its interaction with AXL.

Keywords: AXL; Flaviviruses; Zika virus; fetal endothelial cell; placental barrier.

Fig. 1.

HUVECs are more susceptible to ZIKV infection than to DENV or WNV. (A) HUVECs were infected with Vero 76-produced ZIKV, DENV, or WNV at an MOI of 1. Infection levels, assessed at 24 h postinfection by staining permeabilized cells with the pan-flavivirus antibody 4G2, were normalized to that of ZIKV within each donor. Averages ± SD of three experiments performed in duplicate are shown. ***P < 0.0001. See also Fig. S1. (B) Infection profiles of a representative experiment from A are shown as histograms, where infected cells (colored lines) are compared with mock-infected cells (gray lines). (C) Similar to A, except that cells were fixed and stained with the antibody 4G2 on multiwell plates. (D) The progeny viruses in the supernatants from the experiments in A were quantified by plaque assays in Vero cells. The average titers based on three independent experiments are presented as plaque-forming units per milliliter.

Fig. 2.

AXL is the primary ZIKV entry factor in HUVECs. (A) The cell-surface expression of the indicated proteins (red) or isotypes (gray) was assessed in HUVECs. See also Fig. S2. (B) HUVECs were preincubated with an anti-AXL antibody (AF154) or control IgG, and infected with ZIKV or IAV. Infection levels were normalized to those of cells infected without antibody. (C, Left) Similar to A, except that an anti-MERTK antibody (AF891) was compared with the anti-AXL antibody. Infection levels were normalized as in A. (C, Right) The ability of the antibody to bind MERTK on HUVECs is shown. (D, Left) The efficiency of AXL gene editing via the CRISPR/Cas9 method, directed by an AXL-specific sgRNA in HUVECs (AXL^KO cells; red) and an untargeted sgRNA (control cells; blue), is shown. Cells were stained with anti-AXL antibody (clone 108724). (D, Right) These cells were infected with ZIKV or IAV and infection levels were normalized to those of control cells for each virus. (E, Left) AXL expression was analyzed, using the anti-AXL antibody (clone 108724), in HUVECs transfected with the indicated siRNA. (E, Right) These cells were infected with ZIKV at an MOI of 1, and infection levels were normalized to those of cells transfected without any siRNA. ZIKV was produced in Vero 76 cells and IAV in Madin–Darby canine kidney (MDCK) cells. (B–E) Averages ± SD of three (B and C) or five (D and E) experiments performed in duplicate are shown. **P < 0.001, ***P < 0.0001.

Fig. 3.

ZIKV, but not DENV or WNV, uses AXL efficiently. (A and B) HUVECs, preincubated with 50 nM anti-AXL antibody (AF154) or control IgG (A), or HUVEC AXL^KO and control cells (B), were infected with Vero 76-produced ZIKV, DENV, or WNV at an MOI of 1. The progeny viruses at 24 h postinfection were quantified by plaque assays in Vero cells. Results are expressed as plaque-forming units per milliliter. (C, Left) Expression levels of AXL or DC-SIGN in transduced HEK293T cells are shown. (C, Right) Parental HEK293T-, AXL-, or DC-SIGN–transduced cells were infected with ZIKV, DENV, or WNV at the indicated MOI. Results are presented as percent infected cells. Averages ± SD of three (A and C) or five (B) experiments performed in duplicate are shown. *P < 0.01, **P < 0.001, ***P < 0.0001.

Fig. 4.

Gas6 binds to ZIKV but not to DENV or WNV. (A) HUVECs were preincubated with C-Gas6-Ig or TIM1(AA)-Ig, and infected with Vero 76 cell-produced ZIKV, DENV, or WNV at an MOI of 1. Infection levels were normalized to those of cells infected in the absence of any Ig-fusion protein within each virus. (B) Progeny viruses in the culture supernatants from the experiments in A were quantified by plaque assays in Vero cells. Results are expressed as plaque-forming units per milliliter. (C and D) ZIKV, DENV, or WNV, produced in Vero 76 cells, was incubated with Gas6-Ig, C-Gas6-Ig, or TIM1-Ig and immunoprecipitated using protein A-Sepharose beads. (C) The RNA of the bound viruses was extracted and quantified by RT-qPCR. Binding is represented as fold increases normalized within each virus to that of virus incubated with C-Gas6-Ig. See also Fig. S6. (A–C) Averages ± SD of three experiments performed in duplicate (A and B) or quadruplicate (C) are shown. ***P < 0.0001. (D) Bound viruses were analyzed by Western blot (WB) using antibodies against E protein. A fraction (15%) of the input virus was loaded as a quantity control. A representative experiment of three performed is shown. IP, immunoprecipitation.

Fig. 5.

DENV and WNV produced in C6/36 cells use AXL, whereas those produced in Vero 76 cells do not. HUVECs preincubated with 50 nM anti-AXL (AF154) or control antibody (A and B), or HUVEC AXL^KO or control cells (C and D), were infected at an MOI of 1 with DENV or WNV produced either in Vero 76 or in C6/36 cells. (A and C) Results are presented as percent infected HUVECs. (B and D) Progeny viruses at 24 h postinfection were quantified by plaque assays in Vero cells. Results are expressed as plaque-forming units per milliliter. Averages ± SD of three experiments performed in duplicate are shown. **P < 0.001, ***P < 0.0001.